CN112237755B - Preparation method and preparation device of platelet-rich plasma and prepared platelet-rich plasma - Google Patents
Preparation method and preparation device of platelet-rich plasma and prepared platelet-rich plasma Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D17/00—Separation of liquids, not provided for elsewhere, e.g. by thermal diffusion
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- B01D17/0217—Separation of non-miscible liquids by centrifugal force
Abstract
The invention provides a preparation method of Platelet Rich Plasma (PRP), which comprises the steps of providing a raw material mixed solution, wherein the raw material mixed solution comprises whole blood and an anticoagulant; centrifuging the raw material mixed solution for the first time until the raw material mixed solution is separated into two layers: a first layer and a second layer, the second layer being located in a lower portion of the first layer; extracting the lower 1/4-1/3 of the first layer and the upper 1/4-1/3 of the second layer according to the volume, and mixing the extracted two liquids to obtain a second mixed liquid; and performing secondary centrifugation on the second mixed solution until the second mixed solution is divided into three layers which are sequentially arranged from top to bottom, and taking an intermediate layer to obtain the platelet-rich plasma. The invention also provides a PRP and platelet-rich plasma preparation device prepared by the preparation method. The PRP preparation method provided by the invention can effectively avoid the aggregation of platelets in the preparation process by improving the centrifugal process, and has the advantages of simple and convenient process and easy control.
Description
Technical Field
The invention relates to the field of blood products, in particular to a preparation method and a preparation device of platelet-rich plasma and the prepared platelet-rich plasma.
Background
Platelets are small cytoplasm which is obtained by cracking and removing cytoplasm of mammalian marrow megakaryocyte, have biological activity, and are found to release various growth factors such as transforming growth factor TGF, platelet derived growth factor PDGF, insulin-like growth factor IGF, vascular endothelial growth factor VEGF, epidermal growth factor EGF and the like under the activated state.
TGF has the important functions of promoting mitosis of fibroblast, preosteoblast and vascular endothelial cells, generating, proliferating and differentiating cells, promoting expression of extracellular matrix (ECM collagen, fibrin, etc.) and inhibiting degradation of ECM. PDGF is one of the earliest growth factors in wound surface, and has receptors for PDGF in human body, such as vascular endothelial cells, fibroblasts, macrophages, bone marrow stromal cells, etc., which can make these cells undergo mitotic multiplication, promote the formation of blood vessels, increase the synthesis of collagen, activate the action of macrophages and other growth factors. Macrophages, when activated, have the function of removing necrotic tissue and can release various other growth factors in the second stage, and play a vital role in the repair of the middle and later stages of wounds. VEGF can promote the healing of chronic wounds by inducing or promoting the formation of new blood vessels through autocrine or paracrine, forming a microenvironment favorable for bone regeneration. IGF has the function of promoting cell division and further differentiation, and it has a synergistic effect with PDGF. EGF is a powerful cell division promoting factor and can promote synthesis and deposition of extracellular matrix.
Platelet-rich plasma (PRP) is a platelet-rich plasma product derived from humans and its role in promoting wound healing and tissue regeneration repair is accomplished by the interaction and inter-regulation of the various growth factors described above. The growth factors are immediately adhered to the surface of a target cell membrane after being secreted, so as to activate cell membrane receptors, and the membrane receptors induce intrinsic signal proteins again, so that normal gene sequence expression of cells is stimulated, and the normal healing process is accelerated to release and activate the growth factors, thereby promoting tissue regeneration and repair, such as cartilage injury repair, tendon and ligament injury repair, difficult-to-heal wound repair and the like.
The existing PRP products are mostly prepared by a centrifugal process after blood collection, and the actual platelet content and platelet activity are low. Therefore, there is an urgent need to develop a new PRP preparation method having a higher platelet content.
Disclosure of Invention
In order to overcome the defects in the prior PRP preparation process, the invention aims to provide a preparation method of platelet-rich plasma, which can effectively avoid the aggregation of platelets.
It is another object of the present invention to provide a platelet rich plasma with a higher concentration of enrichment.
It is another object of the present invention to provide a device for preparing platelet rich plasma.
An embodiment of the present invention provides a method for preparing platelet rich plasma, comprising: providing a raw material mixed solution, wherein the raw material mixed solution comprises whole blood and an anticoagulant; the raw material mixed solution is subjected to first centrifugation to separate the raw material mixed solution into two layers: a first layer and a second layer, the second layer being located in a lower portion of the first layer; extracting the lower 1/4-1/3 of the first layer and the upper 1/4-1/3 of the second layer according to the volume, and mixing the extracted two liquids to obtain a second mixed liquid; and performing secondary centrifugation on the second mixed solution to divide the second mixed solution into three layers, and taking an intermediate layer to obtain the platelet-rich plasma.
According to one embodiment of the invention, the intermediate layer comprises a PRP and a leukocyte layer, a 0-3mm erythrocyte layer contiguous with the PRP and leukocyte layer, and a 0-3mm PPP layer contiguous with the PRP and leukocyte layer.
According to an embodiment of the present invention, the height ratio of the first layer to the second layer after the first centrifugation is 40:60 to 60:40.
According to an embodiment of the present invention, the centrifugation conditions for the first centrifugation are: the centrifugal force is 400-800 Xg, and the centrifugal time is 6-30 minutes.
According to an embodiment of the present invention, the centrifugation conditions for the first centrifugation are: the centrifugal force is 550-650 Xg, and the centrifugal time is 6-15 minutes.
According to an embodiment of the present invention, the centrifugation conditions of the second centrifugation are: the centrifugal force is 700-900 Xg, and the centrifugal time is 6-30 minutes.
The invention also provides platelet plasma prepared by the preparation method.
The invention also provides a preparation device of the platelet-rich plasma, which comprises the following steps: a pipetting component and a centrifugation component, the pipetting component being for injecting or withdrawing a liquid; a centrifugation means for subjecting the liquid to centrifugation to prepare the platelet-rich plasma; the centrifugal component comprises at least one centrifugal tube, the centrifugal tube comprises a tube body and a float movably arranged in the tube body, the float is communicated with an inlet of the centrifugal tube, and a mark for marking the position of a liquid interface after centrifugal treatment is arranged on the side wall of the tube body.
According to one embodiment of the invention, the volume between the mark and the bottom of the tube body is 40% -60% of the effective volume of the tube body.
According to one embodiment of the invention, the centrifuge tube comprises a first sealing cover and a second sealing cover which are arranged at two ends of the tube body, the inlet is arranged on the first sealing cover, a channel is arranged on the buoy, and the channel is communicated with the inlet through a hose.
According to one embodiment of the invention, a vent hole is formed in the first sealing cover, and a sterile filter membrane is arranged on the vent hole.
According to one embodiment of the invention, the float is a cylinder, the channel is in a truncated cone shape, the height of the channel is equal to that of the cylinder, the first port of the channel is positioned on the first bottom surface of the cylinder, the second port of the channel is positioned on the second bottom surface of the cylinder, and the diameter of the first port is smaller than that of the second port.
According to an embodiment of the invention, the diameter of the second port is the same as the inner diameter of the bottom surface of the cylinder.
According to one embodiment of the invention, the device comprises a centrifugal tube sleeve comprising a tube, a tube cover and two elastic bodies, which are arranged at both ends of the tube, respectively.
The PRP preparation method provided by the invention can effectively avoid the aggregation of platelets in the preparation process by improving the centrifugal process, and has the advantages of simple and convenient process and easy control. The PRP prepared by the preparation method has high content of platelets, and is suitable for clinical application in multiple fields.
Drawings
FIG. 1 is a microscopic view of platelet rich plasma prepared using the prior art;
FIG. 2 is a schematic view of a centrifuge tube according to one embodiment of the present invention;
FIG. 3 is a schematic view of a centrifuge tube in a centrifuge tube socket according to one embodiment of the present invention;
FIG. 4A is a photograph of the layers of example 1 after the first centrifugation;
fig. 4B is a photograph of the layers after the second centrifugation of example 1.
Detailed Description
Exemplary embodiments that embody features and advantages of the present invention will be described in detail in the following description. It will be understood that the invention is capable of various modifications in various embodiments, all without departing from the scope of the invention, and that the description and illustrations herein are intended to be by way of illustration only and not to be construed as limiting the invention.
The inventors have found that when the centrifugation process or the centrifugation parameter settings are not reasonable, a "coagulation" of the platelets occurs. Referring specifically to FIG. 1, where a is platelet aggregate and b is red blood cell, the "aggregated" platelet aggregate, due to its size, readily enters the red blood cell layer upon centrifugation, resulting in a very low actual platelet content in the PRP product and a decrease in platelet activity in the PRP product.
In order to avoid the above-mentioned "aggregation" of platelets, an embodiment of the present invention further increases the actual platelet content in the PRP product by performing the centrifugation twice and controlling the liquid after the centrifugation within a certain number of layers.
The PRP preparation method provided by the embodiment of the invention adopts a secondary centrifugation process and comprises the following steps: mixing whole blood and anticoagulant, centrifuging for the first time until the whole blood and anticoagulant are separated into two layers, namely a first layer (yellow transparent layer) and a second layer (red turbid layer) from top to bottom, extracting the lower 1/4-1/3 of the yellow transparent layer and the upper 1/4-1/3 of the red turbid layer according to volume, mixing uniformly, centrifuging the obtained mixed solution for the second time until the mixed solution is separated into three layers, namely an anemic platelet plasma (PPP) layer, a platelet-rich plasma PRP (buffy coat) layer and a leukocyte layer from top to bottom, and taking the middle layer as the platelet-rich plasma.
In the invention, the lower 1/4-1/3 of the first layer refers to 1/4-1/3 volume of the first layer liquid positioned at the lower end of the first layer, and the first layer liquid is connected with the upper end of the second layer; the upper 1/4-1/3 of the second layer refers to 1/4-1/3 of the volume of the second layer of liquid positioned at the upper end of the second layer, and is connected with the lower end of the first layer.
In the preparation method of an embodiment of the present invention, the centrifugation degree of the first centrifugation is required to reach a critical node, namely, a process stage in which the leukocyte layer is about to be formed but not yet formed, at which time the blood is separated into only two layers, namely, a yellow transparent layer (upper layer) and a red turbid layer (lower layer).
In one embodiment, the height ratio of the first layer to the second layer after the first centrifugation is 40:60 to 60:40, e.g., 40:40, 40:45, 40:50, 40:60, 50:40, 50:45, 50:60, 55:40, 55:45, 55:50, 60:40, 60:45, 60:50, etc.
In the invention, the node of the first centrifugation can avoid platelet aggregation, and the control of the node is related to the centrifugation speed and time.
In one embodiment, the centrifugation conditions for the first centrifugation are: the centrifugal force is 400-800 Xg, and the centrifugal time is 6-30 minutes; preferably, the centrifugal force is 550-650 Xg, and the centrifugal time is 6-15 minutes; more preferably, the centrifugal force is 605 Xg and the centrifugation time is 10 minutes. Specifically, the centrifugal force of the first centrifugation may be 450×g, 500×g, 600×g, 610×g, 700×g, 750×g, or the like; the centrifugation time may be 8 minutes, 9 minutes, 11 minutes, 12 minutes, etc.
In one embodiment, the mixture of whole blood and anticoagulant is centrifuged at 400 Xg for 6 minutes and separated into two layers.
In one embodiment, the mixture of whole blood and anticoagulant is centrifuged at 400 Xg for 30 minutes and separated into two layers.
In one embodiment, the mixture of whole blood and anticoagulant is centrifuged at 800 Xg for 6 minutes and separated into two layers.
In one embodiment, the mixture of whole blood and anticoagulant is centrifuged at 800 Xg for 30 minutes and separated into two layers.
In one embodiment, the mixture of whole blood and anticoagulant is centrifuged at 550 Xg for 6 minutes and separated into two layers.
In one embodiment, the mixture of whole blood and anticoagulant is centrifuged at 550 Xg for 15 minutes and separated into two layers.
In one embodiment, the mixture of whole blood and anticoagulant is centrifuged at 650 Xg for 6 minutes and separated into two layers.
In one embodiment, the mixture of whole blood and anticoagulant is centrifuged at 650 Xg for 10 minutes and separated into two layers.
In one embodiment, the mixture of whole blood and anticoagulant is centrifuged at 650 Xg for 15 minutes and separated into two layers.
In one embodiment, the mixture of whole blood and anticoagulant is centrifuged at 605 Xg for 6 minutes and separated into two layers.
In one embodiment, the mixture of whole blood and anticoagulant is centrifuged at 605 Xg for 15 minutes and separated into two layers.
According to the invention, through the centrifugal force and the centrifugal time adjusting node, the blood after the first centrifugation can be controlled to be two layers, so that the phenomenon that the activity of platelets is reduced due to aggregation or the yield of platelets is lower due to the fact that the platelets are separated into three layers by adopting a larger centrifugal force or a longer time in the prior art is avoided.
In the preparation method of the PRP, blood is divided into three layers during the second centrifugation, and the three layers are a platelet-poor plasma (PPP) layer, a PRP and a white blood cell (buffy coat) layer, a red blood cell layer and an intermediate layer which are sequentially arranged from top to bottom, so that the PRP prepared by the preparation method of the invention is obtained.
In one embodiment, the intermediate layer includes a PRP and leukocyte (buffy coat) layer.
In one embodiment, the intermediate layer comprises a PRP and a leukocyte layer, a 0-3mm red blood cell layer adjacent to the PRP and the leukocyte layer, and a 0-3mm PPP layer adjacent to the PRP and the leukocyte layer, i.e., the intermediate layer may comprise a small number of PPP layers and/or a small number of red blood cell layers in addition to the PRP and the leukocyte layer.
In one embodiment, the thickness of the red blood cell layer in the intermediate layer may be greater than 0 and less than or equal to 3mm, such as 0.1mm, 0.5mm, 0.8mm, 1mm, 1.5mm, 2mm, 2.5mm, etc.
In one embodiment, the thickness of the PPP layer in the intermediate layer may be greater than 0 and less than or equal to 3mm, such as 0.1mm, 0.5mm, 0.8mm, 1mm, 1.5mm, 2mm, 2.5mm, etc.
In the PRP preparation method provided by the invention, the centrifugation conditions of the second centrifugation are as follows: the centrifugal force is 700-900 Xg, and the centrifugal time is 6-30 minutes; preferably, the centrifugal force is 800-850 Xg, and the centrifugal time is 6-12 minutes; more preferably, the centrifugal force is 823 Xg and the centrifugation time is 8 minutes. Specifically, the centrifugal force of the second centrifugation may be 750×g, 780×g, 820×g, 870×g, or the like; the centrifugation time may be 7 minutes, 9 minutes, 11 minutes, etc.
In the preparation method of the PRP provided by the invention, the whole blood comprises whole blood of human beings or other animals, and the anticoagulant can be of common types and contents and can prevent blood from coagulating; preferably, the anticoagulant is ACD-a anticoagulant in a volume ratio to the venous blood of 15:85 to 10:90, such as 15:85, 15:90, 10:90, etc.
The invention also provides platelet-rich plasma which is prepared by the preparation method according to any one of the technical schemes, wherein the platelet enrichment factor can reach 3.5-14 times of whole blood, such as 5 times, 8 times, 10 times and the like.
The PRP prepared by the invention contains high-content platelets, is very suitable for clinical use, especially orthopedics, sports medical science, stomatology, ophthalmology, neurology and the like, and can play a great role in the fields of beauty treatment, hair growth, chronic wound healing and the like.
Typically, PRP is activated at the time of use, and may be activated by one or any combination of the following:
(1) Near physiological activation: i.e. collagen activation in vivo.
(2) Physical activation: with a physical activator having an elongated hole channel design, PRP is repeatedly pushed through the hole channel and activation is achieved by shear friction.
(3) CaCl is adopted 2 And (5) activating.
(4) Thrombin (including bovine thrombin, autologous thrombin) is used for activation.
(5) thrombin+CaCl 2 And (5) activating.
The PRP preparation method provided by the invention can effectively avoid the aggregation of platelets in the preparation process by improving the centrifugal process, and has the advantages of simple and convenient process and easy control. The PRP prepared by the preparation method has high content of platelets, and is suitable for clinical application in multiple fields.
An embodiment of the present invention provides a device for preparing platelet rich plasma, comprising a pipetting component and a centrifugation component; the liquid transferring component is used for injecting or extracting liquid, and the centrifugal component is used for centrifuging the liquid to prepare platelet-rich plasma; the centrifuge component may include a centrifuge, a centrifuge tube, or the like.
In one embodiment, the pipetting component may be a syringe.
In one embodiment, the centrifuge means comprises at least one centrifuge tube, such as two, which may be a straight single chamber centrifuge tube.
In one embodiment, as shown in fig. 2, the centrifuge tube 10 includes a tube 11, where the tube 11 is a cylindrical body, for example, a cylinder, and an upper cap 12 (a first sealing cap) and a lower cap 13 (a second sealing cap) are respectively disposed at two ends of the tube 11 so that the tube 11 can be in a sealed state.
In one embodiment, the upper cap 12 and the lower cap 13 are connected with the tube 11 by interference fit or by gluing, ultrasonic welding, or the like.
In another embodiment, the upper cap 12, the lower cap 13 and the tube 11 may be integrally formed by injection molding or extrusion molding.
In one embodiment, an inlet 121 is formed in the upper cap 12, the inlet 121 is used to provide an interface for a pipetting device, and an inlet cap may be disposed on the inlet 121 to close the inlet.
In one embodiment, the upper cap 12 is provided with a vent 122, a sterile filter membrane may be disposed in the vent 122 to prevent the contamination source from entering the tube 11, and a hole cover may be further disposed on the vent 122 to open the hole cover during centrifugation.
In one embodiment, the tube 11 is transparent or semitransparent (made of transparent material) to facilitate observation of the liquid state in the tube 11. Marks 15 may be provided on the side wall of the tube 11 to identify the interface position of the liquid in the tube 11 after centrifugal separation or the liquid portion to be extracted, and the marks 15 may be provided in different colors and shapes, and may specifically be one or more mark lines, such as mark lines b1, a, b2 shown in fig. 2.
In one embodiment, the interface location of the first and second layers formed after the first centrifugation is at or near the marker 15 so that the operator will control the critical node of the first centrifugation so that the ratio of the first and second layers after the first centrifugation is within a suitable range.
In one embodiment, the volume between the point 15 and the bottom of the tube is 40% to 60%, such as 45%, 50%, 55%, etc., preferably 45% of the effective volume of the tube. The effective volume of the tube body refers to the volume of liquid which can be contained in the tube body, and the centrifuge tube is filled with the raw material mixed liquid before the first centrifugation in the PRP preparation process, namely the volume of the raw material mixed liquid in the centrifuge tube is generally equal to the effective volume of the tube body.
In one embodiment, the mark 15 may also show a certain floating range, but the upper limit of the floating range is that the volume below the mark 15 (the volume between the mark 15 and the bottom of the tube) is not more than 60% of the effective volume of the tube, and the lower limit of the floating range is that the volume below the mark 15 is not less than 40% of the effective volume of the tube, and in the PRP preparation process, the interface position of the first layer and the second layer should be located in the floating range after the first centrifugation.
In one embodiment, the mark 15 includes a plurality of mark lines, namely mark line a, mark line b1 and mark line b2, wherein the mark line a is located at 45% of the effective volume of the tube body (the volume between the mark line a and the bottom of the tube body is 45% of the effective volume) for identifying the position of the boundary between the first layer and the second layer of liquid after the first centrifugation in the PRP preparation process; the mark line b1 is positioned at 63.3% of the effective volume of the tube body, the mark line b2 is positioned at 30% of the effective volume of the tube body, and a liquid part which forms the second mixed liquid and is to be extracted after the first centrifugal separation is arranged between the mark lines b1 and b2.
In one embodiment, a float 14 is provided in the tube 11, the float 14 communicates with the inlet of the centrifuge tube 10, and the pipetting means can inject the raw material liquid into the tube 11 through the float 14.
In one embodiment, the float 14 is disposed on the wall of the pipe 11 and can slide up and down (along the axial direction of the pipe) along the wall of the pipe 11, and the float 14 can divide the interior of the pipe 11 into an upper cavity and a lower cavity.
In one embodiment, the shape of the float 14 matches that of the tube 11. For example, the tube 11 is a circular tube, the float 14 is a cylinder with a diameter equal to or slightly smaller than the inner diameter of the tube 11, so that it can be tightly attached to the inner wall of the periphery of the tube 11 and slidably disposed in the tube 11. Because the float 14 is closely attached to the pipe wall, liquid below the float 14 can be prevented from entering the cavity above the float 14 to the greatest extent in the process of sliding up and down along the pipe wall.
In one embodiment, a channel 141 is provided in the float 14, the channel 141 being connected to the inlet of the upper cap 12.
In one embodiment, the channel 141 is frustoconical with a bottom opening larger than a top opening, and the channel 141 is connected to the inlet through its top opening, preferably the channel 141 is connected to the inlet through a hose.
In one embodiment, the float 14 is a cylinder; the channel 141 is in a truncated cone shape and is opened along the height direction of the cylinder, and the height of the channel is the same as that of the cylinder, that is, the upper port of the channel 141 is located at the upper bottom surface of the cylinder, the lower port is located at the lower bottom surface of the cylinder, and the diameter of the upper port is smaller than that of the lower port, preferably, the diameter of the lower port of the channel 141 is equal to the inner diameter of the cross section of the cylinder.
In one embodiment, a pipetting device, such as a syringe, may be placed in communication with inlet 121 and may be used to inject liquid into the cavity below float 14 or withdraw liquid from the cavity below float 14 via hose and channel 141, in such a way as to minimize disturbance of the liquid below float 14.
In one embodiment, as shown in FIG. 3, a centrifuge tube sleeve may be provided outside the centrifuge tube 10, the centrifuge tube sleeve comprising a sleeve 21, two sleeve covers 22, and two elastomers 23, the sleeve 21 having a cavity for receiving the centrifuge tube 10. An elastic body 23 is arranged in the sleeve 21, the centrifuge tube 10 can be put into a centrifuge tube sleeve before centrifugation, the sleeve cover 22 is pressed down to fix the centrifuge tube, and the centrifuge tube 10 is positioned between the two elastic bodies 23. During centrifugation and operation, the elastomer 23 can reduce disturbance of deceleration inertia and other external forces to the liquid in the centrifuge tube 10, and maintain stability of the liquid layering interface after centrifugation.
In one embodiment, the two elastic bodies 23 are not connected with the sleeve 21 and the sleeve cover 22, and when in use, the two elastic bodies 23 are clamped between the centrifuge tube 10 and the two bottom surfaces of the sleeve 21 under the pressure of the centrifuge tube 10.
In one embodiment, two elastic bodies 23 are respectively connected to two sleeve covers 22.
The invention will now be described in more detail by way of example with reference to the accompanying drawings, in order to make the features and advantages of the invention more apparent. It should be noted that the examples are intended to understand the concept of the present invention and the scope of the present invention is not limited only to the examples listed herein.
The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1PRP preparation
(1) 10 parts by volume of ACD-A anticoagulant is added into a disposable sterile syringe, a syringe push rod is pushed and pulled to enable the anticoagulant to hang on the inner wall of a syringe sleeve, 90 parts by volume of venous blood of a receiver is extracted, and the blood is uniformly mixed with the anticoagulant.
(2) Opening an inlet cover and a hole cover of the centrifuge tube 10, enabling the lower end of the injector to be in liquid communication with a channel 141 through an inlet 121 of the upper cover 12, and injecting the venous blood containing the anticoagulant into the straight single-chamber centrifuge tube 10 with the buoy 14 through the channel 141; thereafter, the centrifuge tube 10 is placed in the sleeve 21 between the two elastic bodies 23, and the sleeve cover 22 is pressed down to fix the centrifuge tube 10.
(3) Placing the same centrifugal tube after trimming into a low-speed centrifugal machine together for first centrifugation, wherein the specific centrifugation conditions are as follows: the raw material liquid in the centrifuge tube was separated into two layers, namely, a yellow transparent layer (upper layer) and a red turbid layer (lower layer) as shown in fig. 4A, by centrifugal force of 605×g for 10 minutes, and the height ratio of the yellow transparent layer to the red turbid layer was about 55:45, and the interface of the two layers of liquid was located substantially at the mark a in the centrifuge tube wall mark 15 shown in fig. 2.
(4) Extracting the liquid part between the marks b1 and b2, namely the lower third of the yellow transparent layer and the upper third of the red turbid layer, uniformly mixing, and then carrying out secondary centrifugation under the following specific centrifugation conditions: and (3) centrifuging for 8 minutes by 823 Xg centrifugal force, separating the mixed solution in the centrifugal tube into three layers, namely a PPP layer, a PRP layer, a leucocyte layer and a red blood cell layer from top to bottom, extracting a yellow layer of about 2mm on the PRP layer and the leucocyte layer to a red blood cell layer of about 1mm below the leucocyte layer, and uniformly mixing to obtain the PRP capable of avoiding platelet aggregation.
Measurement of platelet concentration
The PRP obtained in example 1, venous blood whole blood and PRP disclosed in the prior art were each tested under the same conditions using a blood cell analyzer, and the related data are shown in Table 1. Among them, the PRP prepared by the prior art was the PRP prepared by the method described with reference to CN 108744608A.
TABLE 1
| Detecting items | Example 1PRP | Whole blood | Existing PRP |
| Platelet concentration (10) 9 personal/L) | >1400 | 164 | 57 |
| Leukocyte concentration (10) 9 personal/L) | 26.91 | 4.8 | 0.5 |
Table 1 shows that the platelet concentration of whole blood is 164X 10 9 The concentration of white blood cells is 4.8X10 per liter 9 and/L. PRP prepared in the prior art had a platelet concentration of 57X 10 9 The concentration of white blood cells is 0.5X10 per liter 9 The average hemoglobin content of red blood cells and the average hemoglobin concentration of red blood cells are far lower than the normal value of the original blood, and the conditions of burst surface (over) appear, which are compared with the observed phenomenon of platelet aggregationAnd consistent. The PRP prepared in example 1 of the present invention reached an over level of platelet concentration, i.e., above 1400X 10 9 The concentration of white blood cells is 26.91 multiplied by 10 per liter 9 and/L. This shows that the PRP prepared in example 1 had a platelet enrichment factor greater than 8.53 compared to whole blood, and thus, example 1 gave a high concentration of PRP.
Growth factor measurement of PRP
According to ELISA, the concentration of the growth factor of PRP subjected to low-temperature freezing prepared in example 1 of the present invention was detected by ELISA detection kit, and Table 2 shows the results of partial detection
TABLE 2 growth factor assay results
| Sequence number | Detecting items | Average absorbance | Average content pg/ml |
| 1 | Human vascular endothelial cell growth factor | 0.09 | 39.88 |
| 2 | Human fibroblast growth factor | 0.13 | 141.54 |
| 3 | Human transforming growth factor beta 1 | 0.09 | 51.77 |
| 4 | Human platelet-derived growth factor | 0.10 | 36.01 |
| 5 | Human insulin-like growth factor | 0.12 | 17.32 |
Example 2
The steps, process conditions, etc. of this example are substantially the same as those of the example, with the main differences: the conditions for the first centrifugation were: centrifugal force of 550 Xg for 10 minutes.
The PRP produced was tested under the same conditions as in example 1: platelet concentration is 1369×10 9 The concentration of white blood cells is 18.6X10 per liter 9 and/L.
Unless otherwise defined, all terms used herein are intended to have the meanings commonly understood by those skilled in the art.
The described embodiments of the present invention are intended to be illustrative only and not to limit the scope of the invention, and various other alternatives, modifications, and improvements may be made by those skilled in the art within the scope of the invention, and therefore the invention is not limited to the above embodiments but only by the claims.
Claims (3)
1. A method of preparing platelet rich plasma comprising:
providing a raw material mixed solution, wherein the raw material mixed solution comprises whole blood and an anticoagulant;
the raw material mixed solution is subjected to first centrifugation to separate the raw material mixed solution into two layers: a first layer and a second layer, the second layer being located in a lower portion of the first layer;
extracting the lower 1/4-1/3 of the first layer and the upper 1/4-1/3 of the second layer according to the volume, and mixing the extracted two liquids to obtain a second mixed liquid; and
performing secondary centrifugation on the second mixed solution to divide the second mixed solution into three layers, and taking an intermediate layer to prepare the platelet-rich plasma;
wherein, the centrifugation condition of the first centrifugation is: the centrifugal force is 550-650 Xg, and the centrifugal time is 6-15 minutes;
the centrifugation conditions of the second centrifugation are as follows: the centrifugal force is 700-900 Xg, and the centrifugal time is 6-30 minutes.
2. The method of claim 1, wherein the intermediate layer comprises a platelet rich plasma and a leukocyte layer, a 0-3mm red blood cell layer contiguous with the platelet rich plasma and the leukocyte layer, and a 0-3mm platelet poor plasma layer contiguous with the platelet rich plasma and the leukocyte layer.
3. The method of claim 1, wherein the ratio of the height of the first layer to the height of the second layer after the first centrifugation is from 40:60 to 60:40.
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